Method for the purification of short nucleic acids

ABSTRACT

Method for the purification of a synthetic oligonucleotide from failure sequences comprising contacting a sample containing the desired oligonucleotide in protected water-soluble form with a hydrophilic adsorbent exhibiting anion exchange groups under conditions permitting binding of said oligonucleotide to said adsorbent, whereafter the adsorbed oligonucleotide is deprotected and separated from failure sequences. The characteristic feature of the method is to use an anion exchange adsorbent that binds the protected oligonucleotide under conditions of high as well as low ionic strength.

TECHNICAL FIELD

Purification of synthetic oligonucleotides from mixtures in which thedesired oligonucleotide contain a hydrophobic protecting group.

The number of nucleotide residues in a synthetic oligonucleotide may inprinciple be any integer >1. According to the synthetic routes employedtoday, the number is often <200 and usually <100.

Synthetic oligonucleotides comprise DNAs, RNAs and analogues containingmodified bases, modified sugars and modified phosphate groups. A certainmodification may be repeated in one, more or all of the nucleotideresidues. A typical modification is thiolation in the backbone, e.g. thephosphate group may have one or two of its oxygen atoms replaced withsulphur (phosphorothioate and phosphorodithioate oligomers,respectively). Another typical modification is alkylation in certainbases, e.g. methylation.

TECHNICAL BACKGROUND AND PRIOR ART

The use of oligonucleotides as drugs will dramatically increase thedemand for large scale production of highly purified syntheticoligonucleotides. Today oligonucleotides are typically synthesized byreacting, step-wise and in a predetermined order, 5'-protectednucleotides, activated at their respective phosphate group, with thedeprotected 5-position in a terminal nucleotide residue of a growingoligonucleotide chain attached to a solid support. The most popularprotecting groups for the 5'-position have been strongly hydrophobicwith preference for 4,4'-dimethoxyphenylmethyl (dimethoxytrityl=DMTr).An alternative is 9-phenylxanthen-9-yl (pixyl=Px). The resulting5-terminal protected oligonucleotides have typically been purified bycombining chromatography on strongly hydrophobic matrixes (reverse phasechromatography=RPC) with anion exchange chromatography (IEX). In commonfor all RPC based purification procedures applied so far have been theuse of water-soluble organic solvents (such as acetonitrile) to elutethe adsorbed oligonucleotide.

A typical procedure for purification of oligonucleotides would, atminimum, include the following steps:

1. Cleavage of the oligonucleotide from the solid support used duringthe synthesis. This is normally done by adding concentrated ammonia(25%) at an elevated temperature for several hours.

2. Removal of ammonia.

3. RPC for removal of failure sequences lacking the protecting group inthe 5-position in terminal nucleotide residues.

4. Deprotection either while the desired full length oligonucleotide isstill adsorbed onto the RPC resin or after elution from the resin.Deprotection is normally carried out by treatment with weak acids.

5. Elution of the oligonucleotides from the RPC column by applying anincreasing concentration of organic solvent, e.g. acetonitrile.

6. Loading of the previously eluted oligonucleotide onto an anionexchange column and subsequent elution by applying an increasingconcentration of an inorganic salt.

7. Concentrating and formulating the desired product.

For the synthesis and purification of oligonucleotides, includinganalogues thereof, see for instance Methods in Molecular Biology 20(1993) (Ed. Agrawal S, Humana Press, Totowa. N.J., U.S.A.).

It has been suggested to run the RPC steps in a column in whichparticulate forms of a strongly hydrophobic matrix and an anion exchangematrix have been intimately mixed (U.S. Pat. No. 4,997,927). Thisapproach demands the use of organic solvents such as acetonitrile anddichloromethane.

DRAWBACKS OF THE PRIOR ART METHODS

The earlier known methods have several drawbacks that make large scaleprocesses difficult and expensive to design. The handling of largeamounts of organic solvents demands explosive safe equipment andtoxicity precautions. The production costs are further increased by therecovery and disposal of organic solvents and by extra costs related tolabour and chromatography equipment for running multi-step processes.

OBJECTS OF THE INVENTION

The objects of the invention is to provide a simplified, safer andcheaper method for the purification of synthetic oligonucleotides.

THE INVENTION

These objects are achieved by applying a sample containing the desiredprotected oligonucleotide in water-soluble form onto a hydrophilicchromatography media (adsorbent) that is substituted with anion exchangegroups and that binds the oligonucleotide under conditions of high aswell as low ionic strength (for instance corresponding to aconcentration of NaCl within the interval 0-3M) under conditionsallowing adsorption of said protected oligonucleotide to said adsorbent.

The property of binding the protected oligonucleotide under conditionsof high as well as low ionic strength means that the adsorbent in spiteof its hydrophilic character also expresses a low but measurablenon-ionic binding that most likely is hydrophobic in nature.

In subsequent steps, the adsorbed protected oligonucleotides aredeprotected and the desired full length oligonucleotide eluted by theuse of aqueous solvents, preferably water, containing the appropriatebuffering components.

Appropriate conditions for the different steps should not cause anysignificant undesired degradation of the oligonucleotide to be purified.

Samples

The sample may be any sample containing the desired oligonucleotide witha hydrophobic protecting group. It may be the crude material coming fromthe synthesis of the oligonucleotide after release from the solid phasematrix. Thus the sample may, in addition to the desired oligonucleotideand reagents added for the release, also contain water-soluble forms offailure oligonucleotides (failure sequences) formed in non-wanted orincomplete reactions during the synthesis. The failure oligonucleotidesmay be in protected and/or unprotected form.

Steps

Adsorption of the protected oligonucleotide to the adsorbent.

The important matter is to provide conditions allowing the non-ionicbinding between the protected oligonucleotide and the adsorbent. Thismeans that at low ion concentration both the protected and unprotectedoligonucleotides may be adsorbed in this step, although clear advantagesare seen in arranging for a selective adsorption of protectedoligonucleotides (i.e. a higher salt concentration). This in turn meansthat the conditions are not critical and crude samples may be applieddirectly more or less without any prepurification steps. Afteradsorption it is preferred to apply a washing step in order to removenon-adsorbed sample constituents including, if present, excess agentsfrom cleavage of the oligonucleotide from the support used during thesynthesis. In case both protected and unprotected oligonucleotides havebeen adsorbed it is advantageous to apply conditions permittingselective desorption of oligonucleotides not carrying the hydrophobicprotecting group, i.e. to increase the salt concentration.

For the adsorption step and subsequent washing steps, the conditions arenormally selected within the intervals:

Ionic strength: In principle the ionic strength may vary within wideranges although washing steps utilizing a high ionic strength in thewashing solutions are favourable for high purity of the end product.Normally ionic strengths corresponding to concentrations of NaCl within0-4M, preferably 0.1-3M or 0.5-3M, are efficient. Suitable salts areNaCl or other inorganic water-soluble salts.

Temperature: Normally within 0°-50° C., with preference for 10°-40° C.

pH: alkaline, that usually means a pH-value within 7-14, with preferencefor 8-12.

Higher salt concentrations will elute non-protected oligonucleotides.

Deprotection. This preferably takes place while the protectedoligonucleotide is in an adsorbed state. The conditions are the same asnormally applied for each respective protecting group, although it ispreferred to keep the conditions so that the formed deprotectedoligonucleotides will remain adsorbed (via anion exchange). Thisnormally means that in case the protecting group is transformed to ahydrophobic compound this latter also will remain adsorbed. Typically,the adsorbent is incubated with a cleavage solution matching theprotecting group in order for the deprotection to take place. Forhydrolytically releasable groups, for instance DMTr, the solution oftencontains a relatively strong organic carboxylic acid, such astrifluoroacetic acid, as the cleavage agent. Potentially also dichloroand trichloro acetic acid may be used. In order to secure that thedeprotected oligonucleotides remains adsorbed the ionic strength isnormally held as low as possible (often below 0.5M). Typically thetemperature and incubation times are selected within the intervals0°-40° C. and 1-60 minutes, respectively, bearing in mind that a lowertemperature requires a longer incubation times.

Elution of deprotected oligonucleotides. This step is performed in theusual manner for the elution of oligonucleotides from hydrophilic anionexchangers. The solutions are aqueous, most preferably water, containingappropriate salts (usually inorganic water-soluble salts, such as NaCl)and buffering components. Most preferably the elution is carried outwith a salt gradient in order to elute the oligonucleotides according tolength. The start and end concentrations as well as the steepness of thegradient will depend on the amount and length of the oligomers to beseparated. Elution may also be performed by stepwise changing the ionicstrength. Normally the ionic strength is within in the interval 0-3M andthe steepness within the interval 5-40 column volumes.

The hydrophobic compound derived from the protecting group, will afterthe elution of the oligonucleotides, remain on the adsorbent. Thecompound may be eluted therefrom by the use of aqueous solutionscontaining a hydrophobic water-soluble cosolvent, for instance a loweralcohol, such as isopropanol and ethanol.

As indicated, application of the appropriate order of adsorption,deprotection and elution will lead to high purification on the selectedhydrophilic anion exchange adsorbent. In case one would select aprotocol in which the adsorbed protected oligonucleotides are desorbedbefore deprotection, this will require extra ion exchange steps in orderto separate the desired full length oligonucleotide from shorter forms.

The adsorbent

Suitable adsorbents are pronouncedly hydrophilic but with a weak butmeasurable ability to bind non-ionically to a modified oligonucleotidecarrying one or more hydrophobic groups. The adsorbent shall show no ora very minor unspecific adsorption of proteins and peptides, such ascytochrome C, ovalbumin and angiotensin, but still has the capacity tobind DMTr-protected oligonucleotides at high ionic strength (forinstance corresponding to a concentration of 0.5-3M NaCl). Structurallythis means that the adsorbent should expose an excess hydrophilicgroups, such as alcoholic OH-, anion exchange groups, oligo- andpolyethylene oxide groups etc., on its surface in comparison tohydrophobic groups.

Examples of hydrophobic groups that may be present are hydrocarbylscomprising aromatic rings, and/or straight, branched or cyclic alkylgroups or chains.

Examples of anion-exchanging groups are amino groups, in particulartertiary groups such as diethylaminoethyl and quarternary amino groups,such as trialkylammoniumalkyl (e.g. trimethylammonium-,triethylammonium- and 2-hydroxyethyldiethylammoniumalkyl),dialkylarylammonium-alkyl (e.g. dimethylaniliniumalkyl) andring-containing ammonium groups (e.g. N-methyl(piperidinium)- andpyridiniumalkyl). The alkyl group linking the nitrogen to the adsorbentmay be a short alkylene chain, such as ethylene. The number of anionexchange groups of the adsorbent is not critical for the method. Anionexchangers having the appropriate degree of nonspecific adsorption andbetween 1-1,000 μmole, preferably 50-1,000 μmole, of anion exchanginggroups per ml media may be used. People within the field will have nodifficulties in finding the appropriate combinations of ion exchangecapacity, sample volume and conditions for adsorption, deprotection andelution keeping in mind the general rules for the invention given above.

Several potential useful adsorbents can be imagined from the scientificand patent literature. For instance chromatography adsorbents made fromwater-insoluble polymers based on crosslinked dextran, polyacrylamide,poly(hydroxyalkyl methacrylate) and other polymethacrylates, celluloseetc. onto which appropriate anion exchange and hydrophobic groups havebeen attached. In some cases the base polymer as such may provide thesufficient hydrophobicity.

The adsorbent may also be built of a strongly hydrophobic base matrix,such as polydivinylbenzene and polystyrene (optionally as copolymerswith each other), polyethylene, polypropylene etc., which matrix hasbeen hydrophilised by being coated with any of the above-mentionedpolymers or derivatized on their surface to exhibit the above-mentionedhydrophilicity and anion exchange groups. In this case the appropriateability to bind non-ionically may originate from the base matrix as suchor from the derivatization.

The adsorbent is normally porous and may be in particle forms (such asbeads) or continuous (monolithic). The particle forms may be used in theform of packed or fluidised beds (expanded beds)

Best mode

At the priority date the most preferred adsorbent was commerciallyavailable under the name of SOURCE® 30Q (Pharmacia Biotech AB, Uppsala,Sweden). According to the manufacturer this media is a rigid, porous,spherical monodisperse anion exchanger. The base matrix is made ofpolystyrene/divinyl benzene beads that have been coated with a layer ofcrosslinked alkylethers containing hydroxyl groups. The anion exchangegroups are of the quarternary type (trimethylammonium) and are attachedto the coating via hydrophilic spacer arms. The ability to bind toprotected oligonucleotides (in particular oligonucleotides havinghydrophobic groups at their 5-terminal position) may derive from thebasic polystyrene/divinyl benzene base matrix or from groups introducedduring the hydrophilization (for instance alkyl ether groups).

According to the best mode of the invention, oligonucleotide analoguesin which at least one oxygen in their phosphate group is replaced withsulphur are purified (phosphorothioate and phosphorodithioateoligomers).

The best mode of the invention is further illustrated in theexperimental section.

EXPERIMENTAL SECTION

The Sample: 35 ml of a 25% ammonia solution containing 4460 opticaldensity units (ODU's) measured at 260 nm, 1 cm path length. Theprocessed oligonucleotide was a phosphorothioate 25-mer, synthesized onan OligoPilot(R) DNA/RNA Synthesiser. The amount of 25-mer correspondingto full length product was 2359 ODU's as estimated from absorbancemeasurements and capillary electrophoresis of the crude material.

The column and the adsorbent: The media SOURCE® 30Q (Pharmacia BiotechAB, Uppsala, Sweden) was packed into a HR16 column (Pharmacia BiotechAB) resulting in a packed bed of 16×110 mm. All chromatographicprocedures were performed on BioPilot(R) (chromatography system,Pharmacia Biotech AB). The following steps were applied in the givenorder:

1. The sample was applied onto the column whereafter the column waswashed with two column volumes (Cv) of 10 mm NaOH followed by two Cv of3.0M NaCl, pH 12, and once again with 2×Cv of 10 mM NaOH. In this stepthe non-dimethoxytritylated failure sequences were washed away.

2. The column was washed with 0.4% trifluoroacetic acid until acidic pHin the eluate, approximately 3×Cv, and then left for 20 minutes toobtain complete cleavage of the DMTr group from the 25-mer. The columnwas then reequillibrated to basic pH with 10 mM NaOH.

3. The 25-mer was then eluted and further purified from shortersequences by applying a linear NaCl gradient from 0.8-1.9M NaCl atconstant pH 12 over 25×Cv. Fractions of 10 ml each were collected andanalysed by capillary electrophoreses. The fractions in which the fulllength product constituted more than 92 % of the oligonucleotides werepooled.

4. The column was finally cleaned by applying 2×Cv of 2M NaCl inisopropanol (30% (w/w)).

Analysis of the pool revealed a yield of 66.37 mg of the product with apurity of 98% as determined by capillary electrophoreses. The overallrecovery was 76%. The complete chromatographic process took less thanthree hours.

I claim:
 1. A method for the purification of a synthetic oligonucleotidefrom failure sequences, comprising:contacting a sample containing adesired protected oligonucleotide in protected water-soluble form with ahydrophilic adsorbent exhibiting anion exchange groups under conditionspermitting binding of said protected oligonucleotide to said adsorbent,wherein said adsorbent binds the protected oligonucleotide underconditions of high as well as low ionic strength; deprotecting theadsorbed protected oligonucleotide while adsorbed; and separating saidadsorbed protected or deprotected oligonucleotide from failuresequences.
 2. The method according to claim 1, wherein the adsorbentafter adsorption of the desired nucleotide is incubated with a solutionthata. comprises a deprotecting agent for cleaving off the protectinggroup and b. has an ionic strength maintaining the deprotectedoligonucleotide adsorbed to the adsorbent; said incubation beingperformed under sufficient time for deprotection.
 3. The methodaccording to claim 2, wherein the desired oligonucleotide is desorbedfrom the adsorbent after deprotection.
 4. The method according to claim3, wherein the desorption of the desired oligonucleotide is carried outwith a salt gradient.
 5. The method of claim 1, wherein said failuresequences are separated from said protected oligonucleotide while saidprotected oligonucleotide is bound to said adsorbent.